Utilize este identificador para referenciar este registo: http://hdl.handle.net/10348/4297
Título: High-resolution melting analysis for bird sexing: a successful
Autor: Morinha, Francisco
Travassos, Paulo
Seixas, Fernanda
Santos, Nuno
Sargo, Roberto
Sousa, Luís
Magalhães, Paula
Cabral, João Alexandre
Bastos, Estela
Palavras-chave: Birds
CHD1 gene
High-resolution melting analysis
Molecular sex identification
Real-time PCR
Data: 2013
Editora: Blackwell Publishing Ltd
Citação: Morinha F. el al, 2014
Resumo: High-resolution melting (HRM) analysis is a very attractive and flexible advanced post-PCR method with high sensitivity/ specificity for simple, fast and cost-effective genotyping based on the detection of specific melting profiles of PCR products. Next generation real-time PCR systems, along with improved saturating DNA-binding dyes, enable the direct acquisition of HRM data after quantitative PCR. Melting behaviour is particularly influenced by the length, nucleotide sequence and GC content of the amplicons. This method is expanding rapidly in several research areas such as human genetics, reproductive biology, microbiology and ecology/conservation of wild populations. Here we have developed a successful HRM protocol for avian sex identification based on the amplification of sexspecific CHD1 fragments. The melting curve patterns allowed efficient sexual differentiation of 111 samples analysed (plucked feathers, muscle tissues, blood and oral cavity epithelial cells) of 14 bird species. In addition, we sequenced the amplified regions of the CHD1 gene and demonstrated the usefulness of this strategy for the genotype discrimination of various amplicons (CHD1Z and CHD1W), which have small size differences, ranging from 2 bp to 44 bp. The established methodology clearly revealed the advantages (e.g. closed-tube system, high sensitivity and rapidity) of a simple HRM assay for accurate sex differentiation of the species under study. The requirements, strengths and limitations of the method are addressed to provide a simple guide for its application in the field of molecular sexing of birds. The high sensitivity and resolution relative to previous real-time PCR methods makes HRM analysis an excellent approach for improving advanced molecular methods for bird sexing.
Revisão por Pares: yes
URI: http://hdl.handle.net/10348/4297
Tipo de Documento: Artigo
Aparece nas colecções:DEBA - Artigo publicado em Revista Científica Indexada

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